A major component of barrier function in stratified squamous epithelia is the cornified cell envelope. This is a multi-component 15 nm thick layer of highly insoluble protein deposited on the inner surface of the plasma membrane of the cells. In the case of the epidermis, a 5 nm thick layer of ceramide lipids (lipid enevlope) is attached to the exterior surface. The insolubility of the protein envelope is due in large part to the crosslinking of the constituent proteins by transglutaminases. We have studied two of these proteins in detail, loricrin and the small proline rich (SPR) families. We have expressed human loricrin in bacteria and used it to characterize its structure, biochemical properties, and crosslinking by epidermal transglutaminases in vitro. Similarly, we have expressed in bacteria several SPR proteins and characterized their biochemical and crosslinking properties in vitro. Solution nmr structural studies on SPR proteins are in progress. Preliminary data suggest the central peptide repeat domains adopt novel 3-loop- like protein folds. We have characterized the differential expression properties of the SPR1 and SPR2 proteins in mouse epidermis and epithelial tissues. Our data suggest the SPR proteins seem to function as crossbridging proteins in the cell envelope structure. We have used controlled proteolysis to dissect apart the cornified cell envelope formed in foreskin epidermis in vivo and in epidermal keratinocyte cultures in vitro. The bulk of the protein envelope consists of loricrin (70-80%( admixed with smaller amounts (2-20%) of SPRs. By alkaline hydrolysis in methanol to remove covalently bound ceramide lipids of foreskin cell envelopes, we have been able to explore the innermost aspects of this structure, corresponding to the initial stages of its assembly. Our data suggest that cell envelope assembly is initiated at the site where keratin filaments and many associated proteins meet desmosomes. We have found that the ceramides of the lipid envelope consist of a heterogeneous population of molecules of varying size, and are covalently attached to involucrin, as well as perhaps other proteins, on the protein envelope.